Complete chloroplast genome and phylogenetic analysis of Lonicera caerulea var. edulis (Caprifoliaceae)

Abstract Lonicera caerulea var. edulis, known as “blue honeysuckle” or “Haskap,” is a deciduous shrub that belongs to the Caprifoliaceae family. Characterized by the high cold hardiness and high quality of fruit, it has become a novel cash crop in cold regions worldwide. The lack of available chloroplast (cp) genome information limits studies of its molecular breeding and phylogeny. Here, the complete cp genome of Lonicera caerulea var. edulis was assembled and characterized for the first time. It was 155,142 bp in length with 38.43% of GC content, including 23,841 bp inverted repeat regions (IRs), an 88,737 bp large single-copy region (LSC), and an 18,723 bp small single-copy region (SSC). A total of 132 genes, including 85 protein-coding genes, 8 rRNA genes, and 39 tRNA genes were annotated. Phylogenetic analysis indicated that L. caerulea var. edulis was closely related to L. tangutica. These data and results provide a valuable resource for the development of breeding tools and genetic diversity studies for L. caerulea.


Introduction
Lonicera caerulea var. edulis (Linn.) Turcz. ex Herd. 1864 (blue honeysuckle) is a deciduous shrub that produces blue-black edible berries with a high concentration of anthocyanins, phenolic acids, and polyphenols (Becker and Szakiel 2019). As a circumpolar species, its tree and flower are hardy to À50 C and À7 C, respectively (Plekhanova 2000, Hummer et al. 2012. Thus, its cultivation and breeding have undergone fast progress during the past two decades in the cold regions of the Northern Hemisphere, especially in Russia, China, and Canada (Huo et al. 2005, Gerbrandt et al. 2017. Despite its importance, the phylogenetic position of L. caerulea in Lonicera is still less clear. It was designated as the only species of subsection Caeruleae in section Isika of the subgenus Chamaecerasus (Rehder 1903, Rehder 1913, Hara 1983, Wu and Hong 2011. Because of the lack of plant material or sequence data of L. caerulea, whether L. caerulea should be classified under Isika is still controversial, and the phylogenetic relationship between subsection Caeruleae and other subsections in section Isika remains unclear (Theis et al. 2008, Nakaji et al. 2015. Due to the genomic characteristics of nonrecombination, high conservation, and uniparental inheritance, the complete chloroplast (cp) genome has been accepted as a powerful tool for phylogenetic studies and molecular breeding (Bi et al. 2018). To date (August 2022), more than 40 versions of Lonicera cp assemblies have been released in GenBank, but L. caerulea has not been included.
Here, the cp genome of L. caerulea var. edulis was assembled and characterized for the first time and a plastome phylogeny of the genus Lonicera was reconstructed, providing useful genetic resources for the molecular breeding of L. caerulea and new insight into the phylogeny of Lonicera species.

Materials
Fresh leaves were obtained from a plant of L. caerulea var.  From the center outward, the first track shows the dispersed repeats, which consist of direct (red arc) and Palindromic (green arc). The second track shows the long tandem repeats as short blue bars. The third track shows the short tandem repeats or microsatellite sequences as short bars with different colors (Black: complex repeat; Green: 1-unit repeat). The small single-copy (SSC), inverted repeat (IRa and IRb), and large single-copy (LSC) regions are shown on the fourth track. The GC content is plotted on the fifth track. The annotated genes are shown on the sixth track (codon usage bias is displayed in the parenthesis) and their functional classifications are color-coded in the bottom left corner. The transcription directions for the inner and outer genes are clockwise and anticlockwise, respectively.

Methods
Total genomic DNA was extracted from 0.1 g of fresh leaves using a modified CTAB method (Doyle and Doyle 1986). A paired-end library with an average insert size of 350 bp was constructed using the MGIEasy PCR-Free DNA Library Prep Set and sequenced on the DNBSEQ TM -T7 platform (MGI Tech Co., Ltd., Shenzhen, China). The raw reads were filtered using SOAPnuke (Chen et al. 2017) with the parameters "-lowQual ¼ 20, -nRate ¼ 0.005, qualRate ¼ 0.5" (Supplementary File 1 and 2). The cp genome was assembled using GetOrganelle (Jin et al. 2020) with a k-mer set of "21, 45, 65, 85, 105" (Supplementary File 3 and Figure S1). The assembled genome was annotated using GeSeq (Tillich et al. 2017) and CPGView (Liu et al. 2022a). The 24 complete Lonicera cp genomes sequences were selected for phylogenetic analysis according to recent studies ( Figure 3). The 8 representative species in Caprifoliaceae and Adoxaceae were selected according to classical taxonomy (Wu and Hong 2011, The Angiosperm Phylogeny Group et al. 2016, Stevens 2017) and recent molecular studies (Theis et al. 2008, Nakaji et al. 2015, Bai et al. 2017, Wang et al. 2021). All of the sequences were aligned using MAFFT v7.505 (Katoh and Standley 2013) and then trimmed using trimAL (Capella-GUTI eRREZ et al. 2009) in the "-automated1" pattern. The maximum likelihood tree was constructed using IQ-TREE 2.2.0.3 with the best-fit model of "TVM þ F þ I þ IþR6" and 1,000 ultrafast bootstraps (UFBoot) replicates for branch support (Minh et al. 2020).
Phylogenetic analysis (Figure 3) showed that the outgroup (Adoxaceae) was located at the base of the tree, and Caprifoliaceae fell into 7 clades with a highly supported topology of (Weigela, (Lonicera, ((Triplostegia, Patrinia), (Kolkwitzia, (Zabelia, Morina))))), which highly supports the molecular taxonomy of Caprifoliaceae . The Lonicera clade is divided into two main clades, namely subgenus Lonicera (L. tragophylla) and subgenus Chamaecerasus (other 24 Lonicera species). However, no hierarchical topology was found among or inside sections Coeloxylosteum and Isika. L. caerulea var. edulis (subsection Caerueae) clustered with L. tangutica (subsection Purpurascentes) in the same branch on the clade of section Isika, indicating their close phylogenetic relationship. These results challenge the subclassification of the subgenus Chamaecerasus (Rehder 1903, Rehder 1913, suggesting that multi-data molecular phylogenetic studies are needed to resolve species relationships and guide taxonomic revisions for the genus Lonicera.

Conclusion
The cp genome characterization and phylogenetic analysis of L. caerulea provide useful genetic resources and new insights for future molecular breeding and phylogenetic studies.

Ethical statement
Appropriate permissions were granted by NEAU before sampling. The initial wild collection followed the "Regulations of the People"s Republic of China on the Protection of Wild Plants guidelines" (http://www.forestry.gov.cn) and complied with permission from the Heilongjiang Forestry and Grassland Bureau (http://lyhcyj.hlj.gov.cn/).

Disclosure statement
No potential conflict of interest was reported by all the coauthors.

Funding
This work was supported by the National Natural Science Foundation of China (32102332), the China Postdoctoral Science Foundation (2021MD703804), the Natural Science Foundation of Heilongjiang Province (LH2021C031) and the "Young Talents" Foundation of NEAU (20QC06).

Data availability statement
The cp assembly in this study is openly available in GenBank of NCBI (https://www.ncbi.nlm.nih.gov/) under accession no. OP345475. The associated BioProject, SRA, and BioSample numbers are PRJNA870700, SRR21133392, and SAMN30383140 respectively.